Feb 01, 2024
Aggregation of human recombinant alpha-synuclein
- 1UCL Institute of Neurology
Protocol Citation: Minee L Choi 2024. Aggregation of human recombinant alpha-synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr35obvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94534
Keywords: ASAPCRN
Abstract
This protocol describes a method of generating aggregation of human recombinant alpha-synuclein.
Humanrecombinant α-Syn Monomeric WTα-Syn is purified from Escherichia coli as
previously described in
CITATION
LINK
https://doi.org/[Hoyer et al., 2002].
Aggregation reactions are carried out using a solution of α-Syn 70 micromolar (µM) in 25 millimolar (mM) Tris buffervsupplemented with 100 millimolar (mM) NaCl, 7.4 (in the presence of 0.01 % volume % NaN3 to prevent bacterial growth).
The buffer is freshly prepared before each experiment and passed through a 0.02 µm
syringe filter (Anotop, Whatman) to remove insoluble contaminants.
Prior to incubation, the reaction mixture is ultra-centrifuged at 90k r.p.m. for 01:00:00 at
4 °C to remove potential seeds.
1h
The supernatant is collected and separated in two fractions: one kept at 4 °C at all
times until use(monomers), and a second incubated in the dark at 37°C 37 °C and 200 rpm , during 07:00:00 - 08:00:00 hours to avoid fibril formation (monomers+oligomers).
15h
α-Syn is always kept in LoBind microcentrifuge tubes (Eppendorf, Hamburg, Germany) to
limit surface adsorption.
Citations
Step 1
Hoyer W, Antony T, Cherny D, Heim G, Jovin TM, Subramaniam V. Dependence of alpha-synuclein aggregate morphology on solution conditions.
https://doi.org/